BMC methods最新文献

{"title":"Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods","authors":"S. Robinson, Mark Samuels, William Jones, Nicolas A. Stewart, Murat Eravci, N. Mazarakis, D.C. Gilbert, Giles Critchley, G. Giamas","doi":"10.1186/s44330-024-00007-2","DOIUrl":"https://doi.org/10.1186/s44330-024-00007-2","url":null,"abstract":"","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141714041","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mini-MEndR: a miniaturized 96-well predictive assay to evaluate muscle stem cell-mediated repair.","authors":"Nitya Gulati, Sadegh Davoudi, Bin Xu, Saifedine T Rjaibi, Erik Jacques, Justin Pham, Amir Fard, Alison P McGuigan, Penney M Gilbert","doi":"10.1186/s44330-024-00005-4","DOIUrl":"10.1186/s44330-024-00005-4","url":null,"abstract":"<p><strong>Background: </strong>Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. To offer greater throughput for functional validation studies, we miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of in vivo muscle endogenous repair.</p><p><strong>Methods: </strong>The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with human myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted within the engineered template. Murine MuSCs are fluorescently labeled, enabling co-evaluation of human and mouse Pax7⁺ cell responses to drug treatments. A regenerative milieu is introduced by injuring the muscle tissue with a myotoxin to initiate endogenous repair \"in a dish\". Phenotypic data is collected at endpoints with a high-content imaging system and is analyzed using ImageJ-based image analysis pipelines.</p><p><strong>Results: </strong>The miniaturized format and modified manufacturing protocol cuts reagent costs in half and hands-on seeding time ~ threefold, while the image analysis pipelines save 40 h of labour. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardizes myotube template quality. In vivo outcomes (enhanced muscle production and Pax7⁺ cell expansion) to a known modulator of MuSC mediated repair (p38/β MAPK inhibition) are recapitulated in the miniaturized culture assay, but only in the presence of stem cells and the regenerative milieu.</p><p><strong>Discussion: </strong>The miniaturized predictive assay offers a simple, scaled platform to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-024-00005-4.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"1 1","pages":"5"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11173370/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}